ABC Transporter Proteins
The proteins of the ATP binding cassette (ABC) transporter superfamily form the largest family of the transmembrane proteins. Most of them are transporter proteins that use the energy from ATP hydrolyses to pump substrates through intra- and extracellular membranes. The transport in most cases is unidirectional; eukaryotes usually efflux substrates from the cytoplasm to the extracellular matrix. The transporter family includes many members (currently 49 human ABC proteins are known) that were classified after phylogenetic analysis into 7 subfamilies (from ABCA-ABCG) [Dean, M, Rzetsky, A és Allikmets R., “The Human ATP-Binding Cassette (ABC) Transporter Superfamily”, Genome Res. 2001 11, 1156-1166]. ABC transporters play a pivotal role in keeping the body healthy and functioning. Their deficiency or malfunction can lead to diseases, like hereditary diseases. The research focusing on their physiological role is still at a relatively early stage.
Within the ABC transporter superfamily several transporters act as a multidrug resistant transporter. Multidrug resistant transporters are transporter proteins that are able to extrude from the cell by active transport a variety of xenobiotics, including drugs. In many cases increase of their activity can lead to resistance against drug therapy. Several members of the transporter family, e.g. MDR1, MRP1 and ABCG2 (MXR/BCRP) have been demonstrated to play a role in the development of drug resistance. The transporters significantly influence the kinetics of drug absorbtion even without any overexpression or activity amplification. As several drugs are substrates of a multidrug transporter, during drug development it is of crucial importance to exclude from the circle of potential drug candidate molecules those ones that can be substrates of such transporters, already at an early phase (early ADME, an acronym for “Absorption, Distribution, Metabolism, Excretion”).
Assays for Studying Membrane Transporters
Membrane transporters are commonly studied in cellular or membrane based assays. To membrane assays the transporters of interests are overexpressed in cellular systems and following expression of the transporters, membrane preparations are isolated from these cells. Insect cell based expression systems, like the insect-Baculovirus system, are frequently applied for protein expression. Insect cells are most frequently derived from the ovary cells of the moth, Spodoptera frugiperda; Sf9 cells are one example.
Thus, the methods for the investigation of the ABC transporters are particularly important; membrane assays are commonly applied for this purpose. Solvo, the applicant itself is also distributor of several membrane assays that are suitable to study interactions between transporters and candidate or lead drugs. Solvo has developed a membrane assay using membranes derived from mammalian cells expressing ABCG2 (MXR-M).
The activity of the transport is usually measured i) either by the rate of ATP consumption ii) or the transport of labeled substrates is monitored. In ATPase assays (i) the transport itself is not measured directly, but via substrate stimulation of ATPase activity, because transported substrates enhance the ATPase activity so they are also referred to as activators. Among transport assays (ii) the determination of vesicular transport is particularly important in the study of transporters. In this assay inside-out vesicles from insect cell membranes are used; and the substrate is transported into the vesicle, where it accumulates and can be detected.
It is of pivotal importance that in vitro models closely mimic the physiological phenotype.
Membranes prepared from human or other mammalian expression systems have been widely used in the field of ABC transporters. Unfortunately, these expression systems usually yield significantly lower expression levels that are insufficient to measure the ATPase activity of the transporter. Moreover, while the transporter to be assayed should be overexpressed, other transporters expressed by the mammalian host cell may contribute to the background or result in a disturbing effect, in particular if the substrate specifities of the transporter proteins overlap.
More robust insect cell expression systems provide usually higher expression rates, and have the clear advantage of being free of other transporters, thus one and only one type of transporter protein can be studied. Assays based on insect cell membrane preparations are particularly preferred as being stable, reliable, easy to handle and quite often several assay formats (substrates stimulated ATPase, vesicular transport and/or nucleotide occlusion) are offered.
Otherwise effective and convenient insect cell and insect cell membrane preparation based assays, however, show certain disturbing differences as compared to mammalian cell based assays, which question their value as a useful and relevant assay system in drug development.
It has been reported for certain ABC transporters, e.g. in case of MDR1 (ABCB1) and ABCG2 as well, that a high basal ATPase activity renders substrate stimulated ATPase assays less sensitive. It has been suggested that the basal ATPase activity, measured in the absence of added substrates, may be due to activation by endogenous substrates, or may reflect a partially uncoupled ATPase activity of the transporter [M. M. Gottesman, T. Fojo, S. E. Bates, Multidrug resistance in cancer: role of ATP-dependent transporters, Nat Rev Cancer 2 (2002) 48-58; G. Szakacs, J. K. Paterson, J. A. Ludwig, C. Booth-Genthe, M. M. Gottesman, Targeting multidrug resistance in cancer, Nat Rev Drug Discov 5 (2006) 219-234]
A surprisingly large difference in the activity of the transporters and the sensitivity for different substrates (sulfasalazine, topotecan, prazosin, mitoxantrone and methotrexate) was observed between the assays based on mammalian or insect cell membranes. Moderated activity and decreased substrate sensitivity for activators/substrates were observed for several transporters expressed in Sf9 membranes. For certain substrates the transporter showed no activity when measured with insect cell derived vesicles.
For example, in case of the wild type ABCG2 transporter, in spite of a pronounced basal ATPase activity, the substrate drug stimulation in the Sf9 cell membranes was relatively small. In contrast, in isolated mammalian cell membranes, ABCG2-ATPase activity could be significantly stimulated by various drug substrates. It was also reported that ATPase activity of the wt ABCG2 protein could not be stimulated by prazosin, a known ABCG2 substrate, when expressed in insect cell membranes [Ishikawa, T., Kasamatsu, S., Hagiwara, Y., Mitomo, H., Kato, R. and Sumino, Y. (2003) Expression and functional characterization of human ABC transporter ABCG2 variants in insect cells. Drug Metab Pharmacokinet 18: 194-202.; Ozvegy, C., Litman, T., Szakacs, G., Nagy, Z., Bates, S., Varadi, A. and Sarkadi, B. (2001) Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. Biochem Biophys Res Commun 285: 111-117.]
Since membrane proteins, when expressed in Sf9 cells are underglycosylated, it was speculated that the loss of glycosylation may result in these alterations of the ABCG2-ATPase activity. In respect of ABCG2 it has been demonstrated that glycosylation has no effect either on its activity, processing, or membrane localization [K. Mohrmann, M. A. van Eijndhoven, A. H. Schinkel, J. H. Schellens, Absence of N-linked glycosylation does not affect plasma membrane localization of breast cancer resistance protein (BCRP/ABCG2), Cancer Chemother Pharmacol 56 (2005) 344-350.; N. K. Diop, C. A. Hrycyna, N-Linked glycosylation of the human ABC transporter ABCG2 on asparagine 596 is not essential for expression, transport activity, or trafficking to the plasma membrane, Biochemistry 44 (2005) 5420-5429.]
So far no direct studies have been conducted to compare the biochemical characteristics of transporters expressed in Sf9 and human systems.
Thus, there is a permanent need in the art for insect cell based and in particular insect cell membrane preparation based ABC transporter assays which have improved physiological relevance and/or improved efficiency.
The present inventors applied a new approach when tried to upload insect cell membranes with cholesterol.
Interaction of Cholesterol with ABC Transporters
As to the interaction of cholesterol with specific ABC transporters, previously it has been suggested that MDR1 (ABCB1, P-glycoprotein or P-gp) is located in the raft/calveola domain and its activity under certain conditions is cholesterol dependent [Troost J, Lindenmaier H, Haefeli W E, Weiss J. “Modulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells” Mol Pharmacol. November 2004; 66(5):1332-9.].
Related to this phenomenon, Kamau S W and coworkers (In Vitro Cell Dev Biol Anim, 2005 41(7) 207-16) have found that cholesterol depletion influences the composition of the membrane lipid, modulates the localization of P-gp and causes its loss of function. The amount of P-gp in the membrane decreased as a result of cholesterol depletion and moved from the “raft” fraction into the fraction that has higher density.
In the U.S. Pat. No. 6,855,812 patent application Hanscom and coworkers describe procedures based on P-glycoprotein. Though they were aware of cholesterol influencing the expression of specific ABC transporters, nonetheless they did not suggest any difference in activities thereof and they were silent about the application of cholesterol to increase activity of ABC transporters or efficiency of the membrane assays.
In fact, MDR1 is the only ABC transporter where the effect of cholesterol on transporter activity has been investigated in detail [Garrigues, A., Escargueil, A. E. and Orlowski, S. (2002) The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane. Proc Natl Acad Sci USA. 99: 10347-10352.] It has been suggested that cholesterol is an ABCB1 substrate, a conclusion challenged lately [Le Goff, W., Settle, M., Greene, D. J., Morton, R. E. and Smith, J. D. (2006) Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis. J Lipid Res 47: 51-58.]
It has been proposed for other transporters too, that at least partially they are localized in raft/caveolar systems. Yunomae and coworkers suggested that the inhibitory effect of dimethyl-beta-cyclodextrin on P-gp and MRP2 function could be attributed to the release of these transporters from the apical membranes into the medium as secondary effects through cholesterol-depletion in caveolae while had no effect on the mRNA levels. Thus, no direct effect of cholesterol to activity was observed [Yunomae K, Arima H, Hirayama F, Uekama K. Involvement of cholesterol in the inhibitory effect of dimethyl-beta-cyclodextrin on P-glycoprotein glycoprotein and MRP2 function in Caco-2 cells. FEBS Lett. Feb. 11, 2003;536(1-3):225-31.].
Among the other membrane transporters primarily the known cholesterol transporter ABCA1 (ABC1) was investigated. Feng, Bo and Tabas, Ira (J. Biol. Chem. 2002 277(48) 43271-43280) have described that the ABCA1 mediated cholesterol and phospholipids efflux was initially induced in cholesterol loaded macrophages, however, later with the accumulation of free (not esterified) cholesterol (which process parallels with the progression of atherosclerotic lesions) it is inhibited and the level of ABCA1 protein also decreases.
Several patent applications [e.g. WO 00/18912 (Schmitz G and Klucken J.), U.S. Pat. No. US 2004/0096851 (Wang N. et al.), U.S. Pat. No. 6,617,122 (Hayden M. R. et al.)] describe the effect of cholesterol on the expression of different ABC transporters. According to our knowledge none of these, however, describe or even mention that cholesterol itself would enhance transport activity.
In US 2004/0185456 the authors (Denefle P. and co.) in example 17 of the description assays are described for the identification of ABC1 protein agonists and antagonists. Vesicles containing the substrates of the protein (e.g. cholesterol and phospoholipid) are applied. No indication has been made however, on the effect of the above mentioned substrates on the activity of ABC1.
Thus, the only observation in the art regarding the effect of cholesterol on the activity of an ABC transporter protein, even if results are somewhat contradictory, concerns MDR1. Nevertheless, in all of these experiments it seems that the authors used mammalian cells or membranes prepared from them. As mentioned earlier, none of the authors refer to the potential application of cholesterol to enhance the activity of ABC transporters, moreover, they do not even suggest the need for such procedure.
In an effort to improve performance of insect cell membrane the present inventors decided to start with the only ABC transporter reported in the art to possibly have a cholesterol sensitive transport capability, i.e. MDR1. They found that membrane cholesterol modulation had only a negligible effect on the activity of the MDR1 multidrug transporter. The conclusion that no improvement of insect cell or insect cell membrane preparation based assays on ABC transporters is possible by cholesterol upload of insect cell membranes was necessarily drawn.
Despite discouraging results the present inventors were unexpectedly able to prepare insect cell membrane preparations and insect cells loaded with cholesterol comprising ABC transporters having increased transport activity in such cholesterol loaded membranes as compared to insect cell membranes comprising the normal level of cholesterol.
In particular, the present inventors unexpectedly found that insect cells or the membrane preparations derived from them, constructed by insect expression systems expressing ABC transporters, like wild type ABCG2 or BSEP (ABCB11), that the activity of the transporter and/or its ligand sensitivity can be increased by increasing the cholesterol content of the membrane. The inventors found that typically cholesterol loading induced activation of the assay systems based on Sf9 cell membranes in both substrate stimulated ATPase and vesicular transport assays.